An alkaline phosphatase (also referred to hereinafter as ALP) is an enzyme used generally as a label in enzyme immunoassay. ALP can be seen in almost all living things ranging from higher animals to bacteria, and in higher animals, there are organ-specific isozymes. It is known that a body fluid such as blood contains endogenous ALP such as liver-derived ALP and bone-derived ALP. Therefore, in the case of immunoassay with ALP as a label, not only an ALP label but also an endogenous ALP derived from a sample reacts with a substrate, thus sometimes failing to attain accurate measurement results.
As a substance useful for suppressing the influence of such endogenous ALP, there is an inhibitor of endogenous ALP. Methods of using an inhibitor of endogenous ALP for suppressing the influence of endogenous ALP are as follows:
U.S. Pat. No. 5,948,630 describes a method of reducing the influence of endogenous ALP, which comprises using a detergent composition containing an inhibitor of human ALP.
EP Publication No. 530490 describes a method which comprises labeling, with ALP, an antibody binding to a cell surface marker such as CD4, and using the ALP-labeled antibody to classify the subtype of a lymphocyte. In this method, a substrate-containing substrate solution/buffer, pH 9.5, and a levamisole-containing cell wash fluid, pH 7.4, are used.
In U.S. Pat. No. 5,948,630, a substrate/wash fluid containing 4-MUP as a substrate and levamisole as an inhibitor of endogenous ALP is used in the Examples. The optimum pH of ALP is alkaline, so in general, reagents including its substrate are set alkaline so that the ALP label can react under the preferable conditions with the substrate. In U.S. Pat. No. 5,948,630, it is described that the pH of the detergent composition is preferably in the range of about 7.0 to 10.0. However, the inhibitor of endogenous ALP such as levamisole is destabilized at alkaline pH. In the detergent composition described in U.S. Pat. No. 5,948,630, therefore, the inhibitor of endogenous ALP cannot be used in a stabilized state in some cases.
In EP Publication No. 530490, cells treated with an ALP-labeled antibody are washed twice with the cell wash fluid, and then the substrate solution/buffer is added to measure staining with ALP activity. Generally, the cell wash fluid is removed from the cells after washing, and thus a reaction solution where the ALP label is reacted with the substrate is substantially free of the cell wash fluid. Accordingly, if endogenous ALP cannot be sufficiently inhibited at the time of washing, there is a possibility that the influence of the endogenous ALP cannot be completely eliminated at the time of the reaction between the ALP label and the substrate.